Glucocorticoids have a broad array of life-sustaining functions and play an important role in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Thus, changes of tissue responsiveness to glucocorticoids may develop pathologic states and influence their disease course. We investigated pathophysiologic mechanism of one of such conditions, familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We analyzed molecular defects of GRL773P, which is recently found as a heterozygotic mutation, replacing leucine at amino acid 773 of GR with proline. This mutant receptor demonstrated reduced transactivation activity due to its inability to form the ligand-dependent transactivation surface and behaved as a dominant negative mutant, thereby the patient developed glucocorticoid resistance even in the hetrozygotic condition. We recently found another two heterozygotic mutations that produce GRD401H and F737L. We will examine their molecular defects as well. To examine biologic defects of pathologic GR mutant receptors in living cells, we examined their motility inside the nucleus in living cells by using the fluorescence recovery after photobleaching (FRAP) method. Motility of the ligand-bound wild type GR is regulated inside the nucleus through its multiple interactions with the chromatin-associated molecules, transcriptional intermediate proteins, target DNAs and the ubiquitine/proteasomal pathway. We found that all of the examined mutant receptors demonstrated increased motility and some of them demonstrated altered responsiveness to a proteasomal inhibitor MG-132, possibly due to loss of one or some of the above-indicated important interactions. In order to find intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings using several bait proteins. We found in the screening using the N-terminal domain of GR as a bait that the guanine nucleotide-binding protein (G) beta and the transforming growth factor beta/bone morphogenetic protein-downstream Smad6 specifically interacted with GR. Both proteins suppressed GR-induced transactivation of glucocorticoid-responsive promoters. The former protein, with its partner molecule Ggamma, co-migrated into the nucleus with GR in response to glucocorticoids, and was attracted to the glucocorticoid response elements (GREs) located in the promoter region of the glucocorticoid-responsive gene, suggesting that attracted Gbeta/gamma interferes with the activity of transcriptional machineries on the GR-bound promoters. Extracellularly administered somatostatin downregulated GR transactivation through the Gbeta/gamma complex. In contrast, Smad6 suppressed GR transcriptional activity both at cellular and animal levels via attracting histone deacetylases and antagonizing to histone acetylation induced by p160 type histone acetyltransferase coactivators. In another yeast two-hybrid screening using the GR DNA-binding domain, we found that SET/TAF-1beta and gas5 interact with this portion of the GR. The former molecule is known as a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex that binds lysine residues of the histones and protects them from acetylation by the histone acetyltransferases. We found that SET/TAF-1beta acts as a negative regulator of GR transcriptional activity and ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through the DBD. We speculate that the SET-CAN gene product may cause glucocorticoid insensitivity in leukemic cells, which harbors this translocation. In contrast, gas5 is none protein-coding mRNA, and is accumulated in growth-arrested cells. In our hands, overexpression of gas5 suppressed GR transcriptional activity, possibly by competing with GR for binding to target DNA sequences. We speculate that gas5 is a growth-related regulator of glucocorticoid action, attenuating the glucocorticoid-induced transcriptional activity in growth-arrested cells. Alternative splicing of the GR gene in exon 9 produces GRbeta in addition to the classic receptor GRalpha. This isoform receptor does not bind glucocorticoids and behaves as a dominant negative inhibitor of GRalpha-mediated transactivation. To explore whether GRbeta exerts distinct, intrinsic biologic effects on the human genome, we examined its impact on endogenous gene expression in human cervical carcinoma HeLa cells, by developing two HeLa cell lines, stably expressing enhanced green fluorescent protein (EGFP)-fused GRbeta or EGFP. We found that overexpression of GRbeta positively or negatively regulated mRNA expression of multiple glucocorticoid-unrelated genes in analyses using the 12K human cDNA microarray. Because most of the GRbeta-regulating genes play important roles in the embryonic development and the cell-to-cell contact, GRbeta might play an important physiologic role in early development, in contrast to GRalpha, the influence of which on gene expression are first evident in the late fetal stage. In addition to above proteins, we also worked on Brx, a Rho type guanine nucleotide exchange factor, which activates Rho family small G proteins by converting them from inactive GDP-bound to active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We found that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to GRE-bound GR. Brx mediated lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. LPA is produced from activated platelets in patients with dysmetabolic syndrome, who frequently develop glucocorticoid hypersensitivity. Thus, Brx may contribute to increased glucocorticoid sensitivity seen in these patients, mediating LPA-induced signal to GR. We found that 14-3-3 proteins were physically associated with the human immunodeficiency virus type 1 accessory protein Vpr and contributed to its cell cycle-arresting activity. 14-3-3 proteins play a significant role in cell cycle progression at several different stages by regulating activities of their partner proteins, such as Cdc25C, Wee1 and Cdk2. They bind to these proteins through phosphorylated serine or threonine residues and modulate their activities by changing their subcellular localization and/or stability. Vpr binds 14-3-3 proteins at their C terminal region and alters the binding ability of the latter to their partner proteins. Thus, Vpr facilitates association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status, promoting cell cycle arrest at the G2/M phase. In addition to the regulation of cell cycle progression, 14-3-3 proteins play a role in the intracellular signaling of insulin, by interacting with key molecules of its signaling cascade, such as insulin receptor substrate 1 (IRS1) and the FoxO subfamily of the forkhead transcription factors. We focused on FoxOs, which function as negative transcription factors of the insulin-signaling pathway. Unphosphorylated, nuclear FoxOs are transcriptionally active, while phosphorylated FoxOs generated through exposure of cells to insulin is segregated into the cytoplasm via binding to 14-3-3, and is thus inactive. We found that Vpr was associated with 14-3-3 and inhibited the latter's interaction with the FoxOs, even though 14-3-3-binding sites are created by insulin. Moreover, we found that Vpr antagonized insulin-induced suppression of the mRNA expression of several insulin/FoxO-target molecules. These results indicate that Vpr may participate in the development of lipodystrophy syndrome/metabolic disturbance frequently observed in AIDS patients through modification of FoxO activity.